Genetic (mutational) and biochemical analyses will be combined in the investigation of mechanisms of promoter site selection and transcription initiation in a well-defined prokaryotic system. Two promoters, the PRM and P'R promoters of bacteriophage lambda, will be studied. The specific aims of the proposed research are: (1) Isolation of new mutations in PRM. These will include reversions (to prm ion) of prm- mutations already isolated and reversions (to prm-) of the prmup-1 mutation (Meyer et al., 1980). (2) Determination of DNA sequence changes associated with the newly-isolated mutations. (3) Investigation of the effects of these mutations and those already isolated on discrete steps in transcription initiation. (4) Isolation of similar mutations in P'R; this will be facilitated by construction of a lambda derivative (cloning vector) into which a fusion of P'R and LacZ has been inserted. (5) Biochemical analyses of mutations in P'R to determine which aspects of transcription initiation are altered. (6) Use of mutants defective in P'R-directed LacZ gene expression to investigate the role of 6S RNA and the lambda Q protein in expression of lambda late genes. These studies can be expected to contribute to an understanding of regulatory processes in prokaryotes, including an understanding of ways that the frequency of transcription initiation can be altered by changes in promoter sequence. In addition the proposed research should help elucidate the nature of DNA sequence elements required for interaction with a complex protein, in this case, E. coli RNA polymerase holoenzyme.